Linking the conformational landscape to enzymatic function through functional site distant mutations

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An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent "$$). catalytic rate (𝑘!"# This observation is where the major point of contention has developed: do "$$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘!"#) or 𝑘!"# will further explore how the modulation of the conformational landscape can indeed fine-tune "$$ without 𝑘!"# impacting 𝑘!"# and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), "$$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘!"# type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.